Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, and is responsible for over 8,000 deaths in the United States each year. The number of HNSCC cases caused by human papilloma virus (HPV) is on the rise, especially in patients 40-65 years old. HPV is currently associated with 80% of oropharyngeal cancers and 5-10% of HNSCC cases from other sub-sites. High-risk HPV strains, most commonly HPV16 and HPV18, cause the preponderance of HPV-positive HNSCC. Current data suggests that in HNSCC, HPV16 limits immune detection by preventing surface presentation of the major histocompatibility complex class I (MHC-I). In HPV-positive HNSCC, MHC-I surface presentation level is associated with the cytotoxic T cell-mediated anti-tumor immune response and thus the identification of strategies to prevent HPV-mediated MHC-I repression may have a significant therapeutic benefit for patients receiving immunotherapies. There some data that suggest E7 negatively regulates transcription of MHC-I constituent genes; however, the detailed mechanism of E7-mediated MHC-I repression is poorly characterized. Here, I have generated two E7-overexpressing HPV-negative HNSCC cell lines and confirmed that E7 protein downregulates transcription of MHC-I. We have developed systematic and logical approaches including CRISPR/CAS9 profiling that we propose to leverage to characterize the molecular mechanism(s) by which E7- represses MHC-I in these models, and HPV+ HNSCC models. Further, we have developed a 3D organoid-based co-culture assay in which these models are co-cultured with patient-matched peripheral blood mononuclear cells (PBMCs) to test the functional effects of de-repression of MHC-I expression. My central hypothesis is that identification of the molecular mechanisms by which HPV16_E7 diminish the expression of MHC-I will lead to the advancement of therapeutic strategies that enhance tumor cell recognition by activated T- cells. I will address this hypothesis through the following aims: 1) Detail the molecular mechanism(s) of MHC locus repression by HPV16_E7 in HNSCC, and 2) Qualify HPV16_E7-dependent MHC class I regulatory pathways in HNSCC. My long-term goal is to develop new therapeutic approaches that improve the overall survival of HPV+ patients, and in doing so, I hope to characterize the specific mechanisms by which HPV16_E7 can prevent MHC-I surface expression in HPV-positive HNSCC.